Microdevice for fusing cells

ABSTRACT

A microdevice for fusing cells including: a microchannel layer including a main microchannel and a plurality of sub-microchannels branched from one end of the main microchannel; a plurality of first electrodes formed on one side of the main microchannel; a plurality of second electrodes formed on the other side of the main microchannel and each second electrode facing the each of the first electrodes; a thin film disposed on the microchannel layer and covering the main microchannel; an upper cover including an air inflow passage for connecting a top of the thin film and the outside of the microdevice; and a power supply unit for applying voltage to the plurality of first and second electrodes.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application claims the benefit of Korean Patent Application No.10-2011-0101882, Oct. 6, 2011, in the Korean Intellectual PropertyOffice, the disclosure of which is incorporated herein in its entiretyby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a microdevice for fusing cells forelectrofusion, which manufactures a desired fused cell at highefficiency.

2. Description of the Related Art

Cell fusion is a method of preparing a hybrid cell by artificiallyfusing two cells in different types. The cell fusion may be performed byusing chemicals or an electric pulse. Here, combining two cells indifferent types by porating a cell membrane via an electric pulse isreferred to as electrofusion.

There are mainly four continuous phases in the electrofusion:dielectrophoresis-based cell alignment, reversible electroporation,reconstruction of cytomembrane, and karyon fusion. Generally, thedielectrophoresis-based cell alignment needs a sinusoidal alternatingcurrent (AC) electric field (intensity: 100 to 300 V/cm) to exert apositive dielectrophoretic (DEP) force on the cells. In addition, ahigh-strength DC electric pulse signal series is required in thereversible electroporation (intensity: 1 to 10 kV/cm, pulse width: 10 to50 μs).

Plate electrodes are usually used in a conventional cell electrofusiondevice. In general, a distance between two plate electrodes is equal toor above 1 cm, and as a result, an expensive generator is required toobtain high-strength electric pluses. Moreover, an electric fieldgenerated between the plate electrodes is uniform, and thusprobabilities of occurrence of reversible electroporation andelectrofusion of aligned cells are equal. Thus, a probability ofoccurrence of unwanted multi-cell electrofusion in the conventional cellelectrofusion device is relatively high.

In order to increase pairing precision, fusion efficiency,multi-function integration, and a degree of automation, a microelectromechanical system (MEMS) and microfluidic technology have beenused to develop microchips for electrofusion. Microstructures in thesemicrochips have a similar scale as cells (5 to 50 μm), and thus usefulin more precise cell manipulation. Also, owing to a short distancebetween two microelectrodes, a high electric field required for cellfusion may be generated even with a low voltage, and thus difficultiesof power supply and high manufacturing costs may be reduced.

However, in a conventional microfluidic device, an average cell fusionefficiency is about 40%, which is higher than a general chemical fusingmethod (use polyethylene glycol (PEG), less than 5%) and a conventionalelectrofusion method (less than or equal to 12%), but a probability offorming desired cell-cell twins is only from 42 to 68%. Accordingly,fusion efficiency of total cells is about 40%×42-68%, i.e., 16 to 30%.In other words, when a cell A and a cell B are to be fused, undesiredhybrid products, such as AA, ABB, AABB, AAB, and BB, may be excessivelyobtained instead of AB.

Accordingly, a new microfluidic chip for fusing desired cells at higherefficiency is required to be developed.

SUMMARY OF THE INVENTION

The present invention provides a microdevice for fusing cells, whereincells to be fused are effectively fused in a one-to-one manner.

According to an aspect of the present invention, there is provided amicrodevice for fusing cells, the microdevice including: a microchannellayer including a main microchannel and a plurality of sub-microchannelsbranched from one end of the main microchannel, wherein an outlet holeis formed at the other end of the main microchannel and a first cellinlet hole and a second cell inlet hole are respectively formed at endsof each of the plurality of sub-microchannels; a plurality of firstelectrodes formed on one side of the main microchannel; a plurality ofsecond electrodes formed on the other side of the main microchannel andeach second electrode facing the each of the first electrodes; a thinfilm disposed on the microchannel layer and covering the mainmicrochannel; an upper cover including an air inflow passage forconnecting a top of the thin film and the outside of the microdevice;and a power supply unit for applying voltage to the plurality of firstelectrodes and the plurality of second electrodes.

According to another aspect of the present invention, there is provideda method of fusing cells, the method including: providing themicrodevice; bending a thin film toward a main microchannel covered bythe thin film by injecting air to a top of the thin film through an airinflow passage of a top cover; injecting first cells and second cellsinto respective inlet holes, and flowing the first and second cellsthrough a sub-microchannel to the main microchannel; applying analternating current (AC) voltage between a first electrode and a secondelectrode such that the injected first and second cells are aligned inthe main microchannel according to a dielectrophoresis; performingelectroporation on the aligned first and second cells by applying directcurrent (DC) pulses between the first electrode and the secondelectrode; applying a quasi-damping AC voltage between the firstelectrode and the second electrode such that the electroporated firstand second cells are fused by being adjacently disposed to each otheraccording to a dielectrophoresis; relaxing the deformed thin film byreleasing the air; and obtaining the fused first and second cellsthrough an outlet hole.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features and advantages of the present inventionwill become more apparent by describing in detail exemplary embodimentsthereof with reference to the attached drawings in which:

FIG. 1 is a perspective view of a microdevice for fusing cells,according to an embodiment of the present invention;

FIG. 2 is an exploded perspective view of a microdevice for fusingcells, according to an embodiment of the present invention;

FIG. 3 is an exploded perspective view of a lower portion, a thin film,and an upper cover of a microdevice for fusing cells, according to anembodiment of the present invention;

FIG. 4 is a perspective view of the lower portion according to anembodiment of the present invention;

FIG. 5 is a perspective view of a substrate according to an embodimentof the present invention;

FIG. 6 is a perspective view of a microchannel layer according to anembodiment of the present invention;

FIG. 7 is a perspective view of a structure of an electrode according toan embodiment of the present invention;

FIG. 8 is a perspective view of a structure of a thin film according toan embodiment of the present invention;

FIG. 9 is a perspective view of a structure of the upper cover accordingto an embodiment of the present invention; and

FIGS. 10A through 10F are schematic internal cross-sectional views fordescribing operations of a microdevice for fusing cells.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, embodiments of the present invention will be described indetail. The invention may, however, be embodied in many different formsand should not be construed as being limited to the embodiments setforth herein; rather, these embodiments are provided so that thisdisclosure will be thorough and complete, and will fully convey theconcept of the invention to those skilled in the art.

The present invention will be described more fully with reference to theaccompanying drawings.

FIG. 1 is a perspective view of a microdevice for fusing cells,according to an embodiment of the present invention, and FIG. 2 is anexploded perspective view of the microdevice. For convenience ofillustration, a power supply unit connected between first and secondelectrodes is not shown, but the power supply unit would have beenobvious to one of ordinary skill in the art.

An embodiment of the present invention provides a microdevice for fusingcells, the microdevice including: a microchannel layer 11 including amain microchannel 111 and a plurality of sub-microchannels branched fromone end of the main microchannel, wherein an outlet hole 114 is formedat the other end of the main microchannel and a first cell inlet hole112 and a second cell inlet hole 113 are respectively formed at ends ofeach of the plurality of sub-microchannels; a plurality of firstelectrodes 121 formed on one side of the main microchannel; a pluralityof second electrodes 122 formed on the other side of the mainmicrochannel and each second electrode facing the each of the firstelectrodes; a thin film 20 disposed on the microchannel layer andcovering the main microchannel; an upper cover 30 including an airinflow passage 31 for connecting a top of the thin film and the outsideof the microdevice; and a power supply unit for applying voltage to theplurality of first electrodes and the plurality of second electrodes.

According to the current embodiment of the present invention, themicrodevice includes a lower portion 10, a thin film 20, and an uppercover 30 as shown in FIG. 3. The lower portion 10 will now be describedin detail.

As shown in FIG. 4, the lower portion includes a microchannel layer 11,a plurality of first electrodes 121 formed on a sidewall of amicrochannel, and a plurality of second electrodes 122 respectivelyfacing the first electrodes 121. Also, a substrate 13 may be furtherdisposed below the microchannel layer 11.

According to an embodiment of the present invention, the microchannellayer 11 may be formed on the substrate 13. The substrate 13 is disposedat the lowest bottom of the microdevice and performs an operation of asupporter as an insulator. A material for forming the substrate 13 isnot limited as long as it is an insulating material, and in detail, thematerial may be silicon, silicon oxide, or glass quartz. A thickness ofthe substrate 13 is not limited as long as it performs the operation asa supporter, and may be equal to or above 400 μm (refer to FIG. 5).

According to an embodiment of the present invention, the microchannellayer 11 includes a main microchannel 111, and a plurality ofsub-microchannels branched from one end of the main microchannel 111. Anoutlet hole 114 may be formed at another end of the main microchannel111, and the first cell inlet hole 112 and the second cell inlet hole113 may be respectively formed at ends of the sub-microchannels as shownin FIG. 6.

The main microchannel 111 and the sub-microchannels are passages wherecells flow through. Two types of cells introduced respectively from thefirst and second cell inlet holes 112 and 113 meet at the mainmicrochannel 111, and the two types of cells are fused in the mainmicrochannel 111. Then, the fused cells are discharged through theoutlet hole 114 formed at the other end of the main microchannel 111.

The microchannel layer 11 may be formed of a material that isbiocompatible, dysoxidative, noncorrosive, and electric resistive. Indetail, Durimide 7510 may be used as the material, but the material isnot limited thereto. Alternatively, a photosensitive material may beused.

The first electrodes 121 are formed on one sidewall and the secondelectrodes 122 facing the first electrodes 121 are formed on the othersidewall of the main microchannel 111 where cells are fused. A voltageis applied to the first and second electrodes 121 and 122 through thepower supply unit, and thus two cells in the main microchannel 111between the first and second electrodes 121 and 122 are fused.

According to an embodiment of the present invention, the firstelectrodes 121 and the second electrodes 122 are respectivelyelectrically connected to holding pads 121 h and 122 h having a shape of

. The holding pads 121 h and 122 h may be manufactured to have a lengthand a height corresponding to those of the main microchannel 111,thereby being fit and fixed to a side of the main microchannel 111 tosurround all of the bottom, side, and top of the main microchannel 111.FIG. 7 illustrates the first electrodes 121 and the second electrodes122, which are respectively electrically connected to the holding pads121 h and 122 h. As shown in FIG. 7, the holding pads 121 h and 122 hmay respectively include pad shapes 121 h′ and 122 h′, which receive apredetermined voltage from the power supply unit.

Each of the first or second electrodes 121 or 122 is formed on thesidewall of the main microchannel 111, and may have a heightcorresponding to a depth of the main microchannel 111 and a widthcorresponding to 1 to 1.5 times of a diameter of a single cell injectedinto the main microchannel 111. The first or second electrodes 121 or122 arranged on the sidewall of the main microchannel 111 may bedisposed at an interval of 3 to 4 times of a diameter of a single cellso that two types of cells in the main microchannel 111 are easily fusedin an one-to-one manner. Accordingly, a repeated structure of anelectrode and a wall of the side of the main microchannel 111 is formedon the side of the main microchannel 111.

A number of electrodes arranged on the side of the main microchannel 111corresponds to a length of the main microchannel 111, i.e., as thelength of the main microchannel 111 increases, the numbers of the firstand second electrodes 121 and 122 increase. Accordingly, the lengths ofthe holding pads 121 h and 122 h surrounding the main microchannel 111are also increased.

The holding pads 121 h and 122 h, the first electrodes 121, and thesecond electrodes 122 may be formed of a material that is biocompatible,dysoxidative, noncorrosive, and electric conductive. Examples of such amaterial include gold, platinum, and titanium, but are not limitedthereto. Thicknesses of the holding pads 121 h and 122 h, the firstelectrodes 121, and the second electrodes 122 may be from 0.2 to 2 μmfor excellent electric conductivity, but are not limited thereto.

According to an embodiment of the present invention, the depth of themain microchannel 111 may be from 17 to 30 μm, but is not limitedthereto. The width of the main microchannel 111 may be equal to or abovea sum of diameters of the first and second cells, and below 1.5 times ofthe sum of the diameters of the first and second cells. Then length ofthe main microchannel 111 is proportional to the number of electrodesdisposed on the side of the main microchannel 111, and may be a littlelonger than the disposed electrodes. The sub-microchannels operate aspassages where cells introduced from each of the first and second cellinlet holes 112 and 113 flow through. A width of the sub-microchannelmay be equal to or above a diameter of a single cell and below 1.5 timesof the diameter of the single cell.

A thin film that is flexible, deformable, and covering the mainmicrochannel 111 is disposed on the microchannel layer 11. The thin filmis not limited as long as it is flexible and deformable, and in detail,may be a polydimethylsiloxane (PDMS) thin film. A thickness of the thinfilm may be from 1 to 15 μm. A length and a width of the thin film maybe sufficient enough to at least cover the main microchannel 111, andcover the entire microchannel layer 11 at maximum. If the thin film hasthe length and width covering the entire microchannel layer 11, holesare formed on the thin film at locations corresponding to the outlethole 114, the first cell inlet hole 112, and the second cell inlet hole113 of the microchannel layer 11. FIG. 8 illustrates the thin filmincluding the holes. Here, diameters of the holes corresponding to theoutlet hole 114, the first cell inlet hole 112, and the second cellinlet hole 113 may be from 1 to 5 mm or 1 to 3 mm, but are not limitedthereto.

The upper cover 30 disposed on the thin film includes an air inflowpassage 31 connecting the top of the thin film to the outside. Accordingto an embodiment of the present invention, the upper cover 30 may have athickness from 50 to 400 μm or 70 to 200 μm, and may be formed of PDMS,but is not limited thereto.

An example of the upper cover 30 is shown in FIG. 9. The upper cover 30is used to cover the microchannel layer 11 covered by the thin film, andincludes holes at locations corresponding to the outlet hole 114, thefirst cell inlet hole 112, and the second cell inlet hole 113 so thatsamples easily flow in and out. Here, diameters of the holescorresponding to the outlet hole 114, the first cell inlet hole 112, andthe second cell inlet hole 113 may be from 1 to 5 mm or 1 to 3 mm, butare not limited thereto.

A channel having a width wider than that of the main microchannel 111 isformed in the upper cover 30, so that air received from the air inflowpassage 31 flows through the channel. A depth of the channel in theupper cover 30 may be from 17 to 30 μm, but is not limited thereto. Whenthe air flows into the upper cover 30, the thin film below the uppercover 30 bends downward according to air pressure, and thus bends towardthe main microchannel 111.

An embodiment of the present invention provides a method of fusingcells, the method including: providing the microdevice; bending a thinfilm toward a main microchannel covered by the thin film by injectingair to a top of the thin film through an air inflow passage of a topcover; injecting first cells and second cells into respective inletholes, and flowing the first and second cells through a sub-microchannelto the main microchannel; applying an AC voltage between a firstelectrode and a second electrode such that the injected first and secondcells are aligned in the main microchannel according to adielectrophoresis; performing electroporation on the aligned first andsecond cells by applying DC pulses between the first electrode and thesecond electrode; applying a quasi-damping AC voltage between the firstelectrode and the second electrode such that the electroporated firstand second cells are fused by being adjacently disposed to each otheraccording to a dielectrophoresis; relaxing the deformed thin film byreleasing the air; and obtaining the fused first and second cellsthrough an outlet hole.

FIGS. 10A through 10F are schematic internal cross-sectional views fordescribing operations of a microdevice for fusing cells. The operationswill now be described with reference to FIGS. 10A through 10F.

First, the microdevice for fusing cells is provided. The microdevice hasa cross-section where a thin film covers a main microchannel disposedbelow the thin film, and an upper cover including a channel wider thanthe main microchannel is disposed on the thin film, as shown in FIG.10A.

When air is injected through an air inflow passage of the upper cover,the thin film below the upper cover bends downward according to airpressure, and thus bends toward the main microchannel as shown in FIG.10B. Accordingly, the inside of the main microchannel is divided intotwo, and thus substantially two microchannels are generated.

Then, a first cell and a second cell are injected respectively throughfirst and second cell inlet holes, and are flowed through the mainmicrochannel. Here, since the thin film divides the main microchannelinto two according to air pressure, and a width of the main microchannelis equal to or above a sum of diameters of the first and second cells,and is below 1.5 times of the sum of the diameters of the first andsecond cells, the first and second cells are not mixed and flow throughthe main microchannel each in a line as shown in FIG. 10C.

Then, an AC voltage (amplitude: 2-20V, frequency: 0.2-3 MHz) is appliedbetween first and second electrodes such that the injected first andsecond cells are aligned in the main microchannel according todielectrophoresis. Due to the thin film bending toward the mainmicrochannel, a strongest electric field is formed at the center of themain microchannel, and thus the first and second cells are adjacentlyarranged at the center according to positive dielectrophoresis as shownin FIG. 10D. Next, electroporation is performed on the first and secondcells that are adjacently arranged by applying DC pulses (amplitude:6-50V, duration: 10-500 μs, interval of two pulses: 0.1-10 s, pulses:1-100) between the first and second electrodes. When the DC pulses areapplied, the first and second cells are reversibly electroporated.

Next, a quasi-damping AC voltage (amplitude: 1-2 V, frequency: 0.2-3MHz, attenuation rate: −0-90%/min) is applied between the first andsecond cells such that the electroporated first and second cells areadjacently disposed and fused according to dielectrophoresis, as shownin FIG. 10E.

Then, as shown in FIG. 10F, the deformed thin film is relaxed bydischarging the injected air, and the fused first and second cells areobtained through an outlet hole. The fused first and second cells may beobtained through the outlet hole by using a syringe pump orelectrophoresis, but a method of obtaining the fused first and secondcells is not limited thereto.

According to the present invention, the first and second cells may existbetween the first and second electrodes each in a line according to thethin film disposed on the microchannel and the air flowing to the thinfilm, and thus the first and second cells having different traits may besmoothly fused in an one-to-one manner when an electric field is appliedbetween the first and second electrodes.

While the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the following claims.

What is claimed is:
 1. A microdevice for fusing cells, the microdevicecomprising: a microchannel layer comprising a main microchannel and aplurality of sub-microchannels branched from one end of the mainmicrochannel, wherein an outlet hole is formed at the other end of themain microchannel and a first cell inlet hole and a second cell inlethole are respectively formed at ends of each of the plurality ofsub-microchannels; a plurality of first electrodes formed on one side ofthe main microchannel; a plurality of second electrodes formed on theother side of the main microchannel and each second electrode facing theeach of the first electrodes; a thin film disposed on the microchannellayer and covering the main microchannel; an upper cover comprising anair inflow passage for connecting a top of the thin film and the outsideof the microdevice; and a power supply unit for applying voltage to theplurality of first electrodes and the plurality of second electrodes. 2.The microdevice of claim 1, wherein each of the plurality of firstelectrodes and each of the plurality of second electrodes areelectrically connected to a holding pad having a shape of

, wherein the holding pad is fit and fixed to a side of the mainmicrochannel.
 3. The microdevice of claim 1, wherein a width of the mainmicrochannel is equal to or above a sum of diameters of a first cell anda second cell, and is below 1.5 times of the sum of the diameters of thefirst and second cells.
 4. The microdevice of claim 1, wherein the thinfilm is flexible and deformable.
 5. The microdevice of claim 1, whereinthe thin film is a polydimethylsiloxane (PDMS) thin film.
 6. A method offusing cells, the method comprising: providing the microdevice of claim1; bending a thin film toward a main microchannel covered by the thinfilm by injecting air to a top of the thin film through an air inflowpassage of a top cover; injecting first cells and second cells intorespective inlet holes, and flowing the first and second cells through asub-microchannel to the main microchannel; applying an alternatingcurrent (AC) voltage between a first electrode and a second electrodesuch that the injected first and second cells are aligned in the mainmicrochannel according to a dielectrophoresis; performingelectroporation on the aligned first and second cells by applying directcurrent (DC) pulses between the first electrode and the secondelectrode; applying a quasi-damping AC voltage between the firstelectrode and the second electrode such that the electroporated firstand second cells are fused by being adjacently disposed to each otheraccording to a dielectrophoresis; relaxing the deformed thin film byreleasing the air; and obtaining the fused first and second cellsthrough an outlet hole.